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The MiSeqDx Platform is a sequencing instrument that measures fluorescence signals of labeled nucleotides through the use of instrument specific reagents and flow cells (MiSeqDx Universal Kit 1.0), imaging hardware, and data analysis software. The MiSeqDx Platform is intended for targeted sequencing of human genomic DNA from peripheral whole blood samples. The MiSeqDx Platform is not intended for whole genome or de novo sequencing.

The MiSeqDx Platform is a high throughput DNA sequence analyzer for clinical use.

The MiSeqDx Platform consists of the MiSeqDx instrument and data analysis software. It is for use with the MiSeqDx Universal Kit 1.0 [MiSeqDx reagent cartridge, MiSeqDx flow cell, SBS Solution (PR2 buffer)] for library preparation and sample indexing (K133136). The end-user inputs extracted genomic DNA to be sequenced and provides the Analyte Specific Reagents (ASRs) to develop a sequencing assay that targets their sequence of interest.

The Illumina MiSeqDx Platform is a high-throughput DNA sequencing instrument. The acceptance criteria and supporting studies are detailed below:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for the MiSeqDx Platform are primarily derived from the special controls stipulated in 21 CFR 862.2265, focusing on accuracy and reproducibility across various genomic features.

2. Sample Sizes Used for the Test Set and Data Provenance

• Accuracy Study 1:
  • Sample Size: 13 unique human genomic DNA samples (from two parents and 11 children). These 13 samples were run in 15 instances (two samples in duplicate). The study queried 24,434 bases across 19 different chromosomes.
  • Data Provenance: The origin of the samples is implied to be human, likely from a well-characterized cohort given the reference to "frequently sequenced by multiple laboratories and sequencing methodologies". The study itself is retrospective/analytical performance data. No specific country of origin is mentioned.
• Accuracy Study 2:
  • Sample Size: 1 sample (NA12878). This sample included 184 amplicons within highly confident reference calls.
  • Data Provenance: The sample (NA12878) is a well-known reference sample established by the National Institutes of Standards and Technology (NIST). This is retrospective data, comparing the device's output to an established reference. No specific country of origin is mentioned.
• Accuracy Study 3:
  • Sample Size: Six samples (implicitly, since the table shows 6 amplicons with 1 "sample" column). The study evaluated "a subset of CFTR clinically significant genetic variations".
  • Data Provenance: These samples were "characterized by bidirectional Sanger sequencing as a reference method." This is retrospective analytical performance data. No specific country of origin is mentioned.
• Reproducibility Study 1:
  • Sample Size: 13 unique human genomic DNA samples similarly to Accuracy Study 1. These samples were run over nine runs, with each run generating results for 15 samples (total of 135 samples run for each amplicon). For lot-to-lot reproducibility, 94 samples and two non-template controls were tested across three lots.
  • Data Provenance: Same as Accuracy Study 1, human samples, retrospective analytical performance data.
• Reproducibility Study 2:
  • Sample Size: Two well-characterized panels of 46 samples each were tested. This resulted in a total of 810 calls per site (46 samples * 2 operators * 3 sites * X amplicons, though the table shows 810 total calls per specific genotype).
  • Data Provenance: Genomic DNA from cell lines with known variants and leukocyte-depleted blood spiked with cell lines with known variants in the CFTR gene. This is retrospective analytical performance data.
• Interfering Substances Study:
  • Sample Size: Eight whole blood samples representing eight unique genotypes. 48 replicates for each interfering substance test.
• DNA Extraction Study:
  • Sample Size: 14 unique blood samples. Total sample size for each extraction method was 168 (14 samples x 2 operators/extraction method x 3 runs/operator x 2 replicates/extracted gDNA sample).
• DNA Input Study:
  • Sample Size: 14 representative DNA samples. Tested in duplicate at 9 DNA input levels. For individual input levels (1250 ng, 250 ng, 100 ng), 4 samples x 20 replicates (80 samples). For 25 ng, 14 samples x 20 replicates (280 samples).
• Sample Indexing Study:
  • Sample Size: 8 unique DNA samples tested with 96 different indexing primer combinations.
• Specimen Storage/Freeze-Thaw Studies:
  • Sample Size: Six K2EDTA anti-coagulated blood samples (divided into 6 aliquots each) for storage. 15 DNA samples for freeze-thaw.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

The documentation does not explicitly state the "number of experts" or their specific qualifications (e.g., "radiologist with 10 years of experience") for establishing ground truth. Instead, it refers to:

• "Well-characterized composite reference information": This reference database was "derived from the combination of multiple sequencing methodologies, publicly available data, and hereditary information." This implies a consensus approach from various robust sources rather than individual expert adjudication.
• Highly confident genotype established for NA12878 by the National Institutes of Standards and Technology (NIST): NIST reference materials are developed through extensive, rigorous characterization by specialized scientific teams, often involving multiple technologies and consensus building.
• "Bidirectional Sanger sequencing as a reference method": Sanger sequencing is a gold standard for sequence verification. The interpretation of Sanger data typically involves trained personnel (e.g., geneticists, molecular biologists).
• For the reproducibility study, the reference was also "well characterized reference database."

4. Adjudication Method for the Test Set

The concept of "adjudication method" (like 2+1 or 3+1) is typically associated with human reviewer discrepancies, often in image-based diagnostics. For genomic sequencing, ground truth is established using orthogonal, highly accurate sequencing technologies and/or composite reference databases. Therefore, traditional "adjudication" by multiple human experts in the sense of reconciling differing interpretations is not directly applicable here. The ground truth generation methods inherently build in a form of "adjudication" or consensus through:

• Composite Reference Information: Combining data from multiple sequencing methods, public databases, and hereditary information.
• NIST Highly Confident Genotype: A highly rigorous, multi-method approach by a standards organization.
• Bidirectional Sanger Sequencing: Often considered a definitive (gold standard) method, interpretation relies on the inherent reliability of the technology.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. MRMC studies are typically performed in diagnostic imaging to assess the impact of AI on human reader performance. The MiSeqDx Platform is an instrument for high-throughput DNA sequencing, and its performance is evaluated directly against established genomic references, not through human interpretation of complex data that could be aided by AI.

6. Standalone (Algorithm Only Without Human-in-the-Loop) Performance

Yes, the studies entirely describe the standalone performance of the MiSeqDx Platform (instrument and its integrated software for data analysis, base calling, and variant calling). The performance metrics (accuracy, reproducibility, call rate) are reported for the system operating without human interpretation influencing the primary result generation. Human intervention is limited to sample preparation and initiating runs, not in the sequence interpretation itself by the device. The MiSeqDx Reporter software performs de-multiplexing and FASTQ file generation "without user intervention".

7. Type of Ground Truth Used

The ground truth used was primarily:

• Well-characterized composite reference information: This involved combining data from multiple sequencing methodologies, publicly available data, and hereditary information.
• Highly confident genotype established by the National Institutes of Standards and Technology (NIST): A gold standard reference.
• Bidirectional Sanger sequencing data: Another gold standard for sequence verification.
• PCR assay (for deletions): A molecular biology technique used to confirm the presence or absence of specific sequences.
• Human genome reference sequence build 19 (GRCh37/hg19): Used for non-variant base comparisons.

8. Sample Size for the Training Set

The document does not explicitly mention a "training set" in the context of machine learning or AI algorithms. The MiSeqDx is a sequencing platform, and its "software" (RTA and MiSeqDx Reporter) performs base calling, de-multiplexing, sequence alignment, and variant calling. If there were any machine learning components in these software modules, the training data used to develop them are not detailed in this document. The studies described are validation studies of the finalized device's performance.

9. How the Ground Truth for the Training Set Was Established

As no specific "training set" for an AI or machine learning algorithm is identified or discussed, the method for establishing its ground truth is not provided. The described ground truth methods (composite reference, NIST, Sanger) are for the test/validation sets used to assess the final device performance.

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